Positive Control Experiment Research Articles

A common stay-on-goal mechanism in anterior cingulate cortex for information and effort choices.

Humans and non-humans alike often make choices to gain information, even when the information cannot be used to change the outcome. Prior research has shown the anterior cingulate cortex (ACC) is important for evaluating options involving reward-predictive information. Here we studied the role of ACC in information choices using optical inhibition to evaluate the contribution of this region during specific epochs of decision making. Rats could choose between an uninformative option followed by a cue that predicted reward 50% of the time vs. a fully informative option that signaled outcomes with certainty, but was rewarded only 20% of the time. Reward seeking during the informative S+ cue decreased following ACC inhibition, indicating a causal contribution of this region in supporting reward expectation to a cue signaling reward with certainty. Separately in a positive control experiment and in support of a known role for this region in sustaining high-effort behavior for preferred rewards, we observed reduced lever presses and lower breakpoints in effort choices following ACC inhibition. The lack of changes in reward latencies in both types of decisions indicate the motivational value of rewards remained intact, revealing instead a common role for ACC in maintaining persistence toward certain and valuable rewards.

A Practical In Silico Method for Predicting Compound Brain Concentration-Time Profiles: Combination of PK Modeling and Machine Learning.

Given the aging populations in advanced countries globally, many pharmaceutical companies have focused on developing central nervous system (CNS) drugs. However, due to the blood-brain barrier, drugs do not easily reach the target area in the brain. Although conventional screening methods for drug discovery involve the measurement of (unbound fraction of drug) brain-to-plasma partition coefficients, it is difficult to consider nonequilibrium between plasma and brain compound concentration-time profiles. To truly understand the pharmacokinetics/pharmacodynamics of CNS drugs, compound concentration-time profiles in the brain are necessary; however, such analyses are costly and time-consuming and require a significant number of animals. Therefore, in this study, we attempted to develop an in silico prediction method that does not require a large amount of experimental data by combining modeling and simulation (M&S) with machine learning (ML). First, we constructed a hybrid model linking plasma concentration-time profile to the brain compartment that takes into account the transit time and brain distribution of each compound. Using mouse plasma and brain time experimental values for 103 compounds, we determined the brain kinetic parameters of the hybrid model for each compound; this case was defined as scenario I (a positive control experiment) and included the full brain concentration-time profile data. Next, we built an ML model using chemical structure descriptors as explanatory variables and rate parameters as the target variable, and we then input the predicted values from 5-fold cross-validation (CV) into the hybrid model; this case was defined as scenario II, in which no brain compound concentration-time profile data exist. Finally, for scenario III, assuming that the brain concentration is obtained at only one time point, we used the brain kinetic parameters from the result of the 5-fold CV in scenario II as the initial values for the hybrid model and performed parameter refitting against the observed brain concentration at that time point. As a result, the RMSE/R2-values of the brain compound concentration-time profiles over time were 0.445/0.517 in scenario II and 0.246/0.805 in scenario III, indicating the method provides high accuracy and suggesting that it is a practical method for predicting brain compound concentration-time profiles.

Efficacy of compost made of food wastes with organic amendments for a sustainable and environment friendly agricultural system

Organic amendments in composts are found to be an effective solution for the sustenance of the agriculture system, especially in reducing greenhouse gases. The objective of this research was to develop a compost type made with organic/mineral amendments for environment friendly agriculture. This preliminary study reports the quality of the compost made with different organic/mineral amendments, the performance of different compost types with and without amendments, the potential of organic amendments in reducing greenhouse gas emissions for an environmentally friendly agriculture and the best type of co-compost. Food waste was co-composted with banana peels, egg shells, onion peels and papaya peels individually with a 1:1 ratio; and the quality of the mature compost was measured using the physicochemical parameters. Six seedlings of Capsicum annuum were planted in potting mixtures with a 1:1:1 ratio of co-compost, top soil and sand. The positive control experiment was done with food waste compost and the negative control was with no compost. The performance of the six treatments were evaluated by measuring the plant height, the number of leaves in a plant, number of buds, number of pods and the average weight of pods. CO2 emission of the six treatments were measured by absorbing it to calcium hydroxide. The results showed that the co-compost made with banana peels was the best amendment, with better physicochemical characteristics and performance in growth. None of the treatments gave a significant reduction on the emission of carbon dioxide.

Improvement of Bioethanol Production from Sweet Sorghum Juice under Very High Gravity Fermentation: Effect of Nitrogen, Osmoprotectant, and Aeration

To improve ethanol production fermentation efficiency from sweet sorghum juice under a very high gravity (VHG, 280 g/L of total sugar) condition by Saccharomyces cerevisiae NP01, dried spent yeast (DSY), yeast extract, and glycine concentrations were optimized using an L9 (34) orthogonal array design. The results showed that the order of influence on the ethanol concentration (PE) was yeast extract > glycine > DSY. The optimal nutrient concentrations for ethanol production were determined as follows: yeast extract, 3; DSY, 4; and glycine, 5 g/L. When a verification experiment under the projected optimal conditions was done, the P, ethanol yield (Yp/s), and ethanol productivity (Qp) values were 120.1 g/L, 0.47, and 2.50 g/L·h, respectively. These values were similar to those of the positive control experiment with yeast extract supplementation at 9 g/L. The yeast viability under the optimal condition was higher than that of the control experiment. To improve sugar utilization and ethanol production, aeration at 2.5 vvm for 4 h was applied under the optimal nutrient supplementation. The P, Yp/s, and Qp values were significantly increased to 134.3 g/L, 0.50, and 2.80 g/L·h, respectively.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Airborne exposure to gaseous and particle‐associated organic substances in resin‐based dental materials during restorative procedures

Dental composite dust has been shown to act as a vehicle for methacrylates in vivo/in vitro. The objective of this study was to assess airborne exposure of dental personnel to gaseous and particle-associated organic constituents from resin-based dental materials in a simulated clinic. Sampling of total aerosol fractions and gaseous substances was performed by dental students carrying particle filters and gas sorbents attached to a personal pump during preclinical restorative procedures in phantom models (n=13). Water from the phantoms was sampled. Organic substances were extracted from the sampled water, particle filters, and gas sorbents. Qualitative and quantitative analyses were performed by gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). The methacrylates 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) and the additives camphorquinone (CQ), butylated hydroxytoluene (BHT), and ethyl 4-(dimethylamino)benzoate (DMABEE), were quantified in the gas and particle fractions sampled. A positive-control experiment was conducted. No methacrylates were detected in the gas or particle fractions sampled, whereas strong signals for methacrylates were detected in the positive controls, matching the analysis of the uncured material. In addition, TEGDMA and DMABEE were quantified in the sampled water. Airborne exposure to constituents in resin-based dental materials was below the detection limit. However, the extent of exposure is probably dependent on the procedure, preventive measures, and type of materials used.

Micropropagation of Dendrobium phalaenopsis Orchid Through Overexpression of Embryo Gene AtRKD4

To increase the efficiency of crop production from Dendrobium phalaenopsis orchids, mass propagation has been performed by inducing somatic embryogenesis through Agrobacterium-mediated transformation of the Arabidopsis embryo gene AtRKD4 into orchid protocorm (developing orchid embryo). The three-week-old protocorms of D. phalaenopsis were genetically transformed with T-DNA carrying 35S :: GAL4 :: AtRKD4 :: GR through A. tumefaciens strain EHA 105. The cultures were maintained in VW medium with 10 mg L-1 Hygromycin. Due to the existence of glucocorticoid response element (GR) in the T-DNA construct, the transformed protocorms were transferred into VW medium with the addition of 15 μM Dexamethasone in 6 weeks after transformation to activate the transgene. A total of 12% protocorms has been confirmed for Hyg + by using PCR. The expression of embryo gene AtRKD4 was confirmed by cDNA analysis using AtRKD4 specific primers and Actin primers as a positive control experiment. The expression level of AtRKD4 in 2.5-month-old D. phalaenopsis transformant shoots was 7 times higher than non-transformant plants, and increased to 86 times higher in 8-months, that much higher than that of non-transformant. These results provide an improved method for genetic transformation of D. Phalaenopsis and will (eventually) increase production efficiency in the future.

In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells

A defining characteristic of optic neuropathies, such as glaucoma, is progressive loss of retinal ganglion cells (RGCs). Current clinical tests only provide weak surrogates of RGC loss, but the possibility of optically visualizing RGCs and quantifying their rate of loss could represent a radical advance in the management of optic neuropathies. In this study we injected two different adeno-associated viral (AAV) vector serotypes in the vitreous to enable green fluorescent protein (GFP) labelling of RGCs in wild-type mice for in vivo and non-invasive imaging. GFP-labelled cells were detected by confocal scanning laser ophthalmoscopy 1-week post-injection and plateaued in density at 4 weeks. Immunohistochemical analysis 5-weeks post-injection revealed labelling specificity to RGCs to be significantly higher with the AAV2-DCX-GFP vector compared to the AAV2-CAG-GFP vector. There were no adverse functional or structural effects of the labelling method as determined with electroretinography and optical coherence tomography, respectively. The RGC-specific positive and negative scotopic threshold responses had similar amplitudes between control and experimental eyes, while inner retinal thickness was also unchanged after injection. As a positive control experiment, optic nerve transection resulted in a progressive loss of labelled RGCs. AAV vectors provide strong and long-lasting GFP labelling of RGCs without detectable adverse effects.

R- and S-terbutaline activate large conductance and Ca2 + dependent K+ (BKCa) channel through interacting with β2 and M receptor respectively

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.

Eplerenone does not limit ischemia–reperfusion injury in human myocardial tissue

BackgroundDespite rapid reperfusion, mortality and morbidity in patients with an acute myocardial infarction remain significant. Therefore, novel pharmacological strategies to further limit ischemia–reperfusion (IR) injury are warranted. In animal models of myocardial infarction, mineralocorticoid receptor antagonists potently limit infarct size. In the current study we aimed to translate these findings to the human situation and investigated for the first time in human myocardial tissue whether eplerenone limits IR-injury. MethodsIn 24 patients undergoing elective cardiac surgery, the right atrial appendage was harvested, and two trabeculae were dissected from each appendage and suspended in an organ bath. We induced contraction by electrical field stimulation. Recovery of contractile force after a period of simulated ischemia and reperfusion was used as well-validated endpoint of IR-injury. From each patients, the trabeculae were randomized to either ischemic preconditioning (IP) or no IP (n=12; positive control experiment) or to superfusion with eplerenone (10μM) or vehicle (n=12) in a paired approach. ResultsIP improved recovery from 19.9 (SEM 3.3)% to 26.3 (SEM 4.3)% (p<0.05). During vehicle and eplerenone superfusion, mean recovery of contractile function after simulated ischemia and reperfusion was 45.2 (SEM 5.6)% and 36.5 (SEM 4.1)% (p=0.14). ConclusionEplerenone does not limit IR-injury in human atrial tissue ex vivo. Our results are in sharp contrast to preclinical studies demonstrating cardioprotective effects of mineralocorticoid receptor antagonist. With great interest we await the results of the MINIMISE-STEMI study, in which the effect of MR antagonism on myocardial infarct size in humans is currently under investigation.

The Probable Datum Method (PDM): a technique for estimating the age of origination or extinction of nannoplankton

Accurate interpretation of origination and extinction of fossil species is crucial to answering a variety of questions in paleontology. Fossil datums, the observed age of first or last occurrences, are subject to sampling error as a result of preservation and low abundances near range endpoints. This sampling error can cause local range offset, an age difference between the observed first or last occurrence of a species and its true origination or extinction. Here, we develop and test a new technique, the Probable Datum Method (PDM), that can be used to assess the extent of local range offset for nannofossil species. The PDM estimates the original abundance of a taxon and its probable true age of first or last occurrence. The PDM uses a model in which original abundance is related to count abundance through preservation and the counting process. This model is empirically parameterized, including an experimental determination of false positive and error rates of a nannofossil count. The model is simulated then inverted to estimate likely original abundance and true datum age from count abundance data. We first test the PDM in a positive control experiment with known parameter values. This experiment shows that the PDM is robust and returns known values accurately. Next we apply the method to the origination of nannoplankton after the Cretaceous/Paleogene boundary to test whether first occurrences were synchronous between widely spaced locations. The PDM results suggest that observed diachrony of K/Pg originations cannot be explained by the effects of local range offset; rather, in some cases they indicate truly diachronous first occurrences between localities. Although the technique was developed to analyze nannoplankton ranges, the statistical nature of the PDM, its experimentally derived parameters, and its parsimonious nature should make it applicable to many micropaleontological studies that interpret patterns of origination and extinction.

Brown adipose tissue activity after a high-calorie meal in humans

Studies in rodents have shown that brown adipose tissue (BAT) is activated on food intake, thereby reducing metabolic efficiency. The current study investigated whether a single high-calorie, carbohydrate-rich meal activates BAT in lean human adults. BAT activity was studied in 11 lean adult men [age: 23.6 ± 2.1 y; body mass index (BMI; in kg/m(2)): 22.4 ± 2.1] after consumption of a high-calorie, carbohydrate-rich meal (1622 ± 222 kcal; 78% carbohydrate, 12% protein, 10% fat). BAT activity during 2 h of mild cold exposure served as a positive control experiment. BAT activity was assessed by [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography-computed tomography. Energy expenditure was measured by indirect calorimetry. Postprandial [(18)F]FDG uptake was significantly higher in BAT [1.65 ± 0.99 mean standard uptake value (SUVmean)] than in subcutaneous (0.35 ± 0.15 SUVmean; P < 0.05) and visceral (0.49 ± 0.24 SUVmean; P < 0.05) white adipose tissue and liver (0.95 ± 0.28 SUVmean; P < 0.05). Postprandial BAT activity was lower than cold-induced BAT activity (7.19 ± 2.09 SUVmean). However, postprandial BAT activity may have been underestimated because of high postprandial [(18)F]FDG uptake in skeletal muscle compared with cold (1.36 ± 0.31 compared with 0.59 ± 0.07 SUVmean, P < 0.05), which reduces [(18)F]FDG bioavailability for BAT and other tissues. No direct relation was found between BAT and diet-induced thermogenesis (DIT). Glucose uptake in BAT increases after a meal in humans, which indicates a role for BAT in reducing metabolic efficiency. However, the quantitative contribution of BAT to DIT relative to other tissues, such as skeletal muscle, remains to be investigated. This trial was registered at www.controlled-trials.com as ISRCTN21413505.

Immunotoxic effects of oil sands-derived naphthenic acids to rainbow trout

Naphthenic acids are the major organic constituents in waters impacted by oil sands. To investigate their immunotoxicity, rainbow trout (Oncorhynchus mykiss) were injected with naphthenic acids extracted from aged oil sands tailings water. In two experiments, rainbow trout were injected intraperitoneally with 0, 10, or 100mg/kg of naphthenic acids, and sampled after 5 or 21d. Half of the fish from the 21d exposure were co-exposed to inactivated Aeromonas salmonicida (A.s.) to induce an immune response. A positive control experiment was conducted using an intraperitoneal injection of 100mg/kg of benzo[a]pyrene, a known immune suppressing compound. T-lymphocytes, B-lymphocytes, thrombocytes, and myeloid cells were counted in blood and lymphatic tissue using flow cytometry. In the 5d exposure, there was a reduction in blood leucocytes and spleen thrombocytes at the 100mg/kg dose. However, at 21d, leucocyte populations showed no effects of exposure with the exception that spleen thrombocyte populations increase at the 100mg/kg dose. In the 21d exposure, B- and T-lymphocytes in blood showed a significant Dose×A.s. interaction, indicating stimulated blood cell proliferation due to naphthenic acids alone as well as due to A.s. Naphthenic acid injections did not result in elevated bile fluorescent metabolites or elevated hepatic EROD activity. In contrast to naphthenic acids exposures, as similar dose of benzo[a]pyrene caused a significant decrease in B- and T-lymphocyte absolute counts in blood and relative B-lymphocyte counts in spleen. Results suggest that the naphthenic acids may act via a generally toxic mechanism rather than by specific toxic effects on immune cells.

Absence of evidence is not evidence of absence: Feral pigs as vectors of soil‐borne pathogens

AbstractInvasive soil‐borne pathogens are a major threat to forest ecosystems worldwide. The newly discovered soil pathogen, Phytophthora ‘taxon Agathis’ (PTA), is a serious threat to endemic kauri (Agathis australis: Araucariaceae) in New Zealand. This study examined the potential for feral pigs to act as vectors of PTA. We investigated whether snouts and trotters of feral pigs carry soil contaminated with PTA, and using these results determined the probability that feral pigs act as a vector. We screened the soil on trotters and snouts from 457 pigs for PTA using various baiting techniques and molecular testing. This study detected 19 species of plant pathogens in the soil on pig trotters and snouts, including a different Phytophthora species (Phytophthora cinnamomi). However, no PTA was isolated from the samples. A positive control experiment showed a test sensitivity of 0–3% for the baiting methods and the data obtained were used in a Bayesian probability modelling approach. This showed a posterior probability of 35–90% (dependent on test sensitivity scores and design prevalence) that pigs do vector PTA and estimated that a sample size of over 1000 trotters would be required to prove a negative result. We conclude that feral pigs cannot be ruled out as a vector of soil‐based plant pathogens and that there is still a high probability that feral pigs do vector PTA, despite our negative results. We also highlight the need to develop a more sensitive test for PTA in small soil samples associated with pigs due to unreliable detection rates using the current method.

Potentiation of Helicobacter pylori CagA Protein Virulence through Homodimerization

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.

Phosphorylation of the Human Retinoid X Receptor α at Serine 260 Impairs Coactivator(s) Recruitment and Induces Hormone Resistance to Multiple Ligands

The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.

1103. Augmentation of Immunity Against AAV- Encoded Transgenes by Use of the Genetic Adjuvants C3d3 and CTLA4

Top of pageAbstract AAV transduction enables long-term transgene expression in immuno-competent hosts because of minimal activation of the innate immune system. However, it is possible to elicit transgene-specific immunity but this is dependent upon the antigen and route of immunization. With this view, we are exploiting the unique biology of AAV as a genetic vaccine vector. The cDNA encoding MSP4 (from Plasmodium falciparum), MSP1-19 or MSP4/5 (both from Plasmodium yoelli) were fused with a TPA leader sequence cDNA (for antigen secretion) and cloned downstream of the CMV immediate/early promoter within an AAV provirus (pTRUF-2). AAV genomes were packaged into AAV1 capsids using a helper virus-free system and injected (1012 vg) into the hindlimb muscle (im) of 8|[ndash]|10 week old female Balb/C mice. Antibody responses specific for the transgene were detected against each vector although those generated against MSP1-19 were poor. No protective efficacy was afforded by vaccination with rAAV- MSP4/5 as tested by challenge with P.yoelli in a model where high titre antibodies to MSP4/5 correlates with protection. We concluded that augmentation of transgene-specific immunity is required to confer protection in this model and we have therefore investigated two genetic adjuvant strategies. The first strategy was the genetic fusion of antigen to three tandem copies of C3d, a complement protein, which has been effective at augmenting immunity in other vaccine vector systems. Unexpectedly, rather than augmenting immunity, im injection of rAAV-MSP4/5- C3d3 did not stimulate detectable antibody responses against MSP4/5. This contrasts with the positive control experiment where genetic fusion of C3d3 with hen egg lysozyme (HEL) was shown to augment anti-HEL antibody responses in the context of an rAAV vector. The second strategy tested was genetic fusion of MSP4/5 with Cytotoxic T-Lymphocyte Activation antigen-4 (rAAV-CTLA4- MSP4/5), which is thought to promote immunity by targeting antigen to antigen-presenting cells. Vectors were initially tested over the range 109|[ndash]|1012 vg/injection and responses to rAAV-CTLA4-MSP4/5 were detected in 3/5 animals given 109 vg. This is in contrast to 1010 vg as the minimal amount of rAAV-MSP4/5 vector required to stimulate detectable antibodies. A subsequent study over the range 106|[ndash]|109 vg showed detectable antibody against |[ge]|108 vg rAAV-CTLA4-MSP4/5 indicating that this strategy can augment immunity 100-fold with low dose vector. Antibody responses against rAAV- CTLA4-MSP4/5 peaked earlier than those against rAAV-MSP4/5 in all groups and immunity was not augmented with |[ldquo]|CTLA4- fused|[rdquo]| vector with |[ge]|1011vg. We conclude that it is possible to augment immunity against AAV encoded antigens using the genetic adjuvants C3d3 and CTLA4 in an antigen and adjuvant-dependent manner. These strategies may prove advantageous for the promotion of immunity against other antigens in the context of AAV, particularly where responses are initially poor, and studies are ongoing to determine whether they may provide protection in the murine model of P.yoelli infection.

Human recombinant mutated forms of the mitochondrial COX assembly Sco2 protein differ from wild-type in physical state and copper binding capacity

The human Sco2 protein is a cytochrome c oxidase assembly protein that participates in mitochondrial copper pathway, acting downstream of Cox17 protein. In a previous work, we detected mutations in the human SCO2 gene in three unrelated infants with fatal cardioencephalomyopathy and COX deficiency. In this study, full-length processed recombinant wild-type and two mutated forms of hSco2p (w/t-rhSco2p, E140K-rhSco2p, and S225F-rhSco2p) were produced in bacteria as soluble recombinant peptides for the first time and evaluated for differences in their physical state and ability to bind copper. Our data indicate the following: (a) w/t-rhSco2p and S225F-rhSco2p were found to be in a monomeric form in contrast to E140K-rhSco2p that was in a major non-reducible dimer and a minor monomer form; (b) wild-type and mutated rhSco2p exhibited clear differences in their physical conformational state, as shown by circular dichroism and thermal denaturation analyses; (c) copper binding studies showed that E140K-rhSco2p bound markedly less copper while S225F-rhSco2p more than expected as compared to amount of the copper bound with w/t-rhSco2p. rhCox17p served as positive control experiment. These data indicate that S225F and E140K mutations found in the SCO2 gene derived from patients alter the physical conformational state of encoded hSco2p that may disturb the normal copper transport pathway in mitochondria. These findings are valuable for understanding the molecular basis of fatal cardioencephalomyopathy and COX deficiency and for designing appropriate pharmacological interventions.

The Effects of Dietary Myoinositol on Lung Tumor Development in Tobacco Smoke-Exposed Mice

The purpose of these experiments was to investigate whether a diet containing myoinositol could prevent the development of tobacco smoke-induced lung tumors in strain A/J mice. In a positive control experiment, 1% and 3% of myoinositol in AIN-93 diet reduced the development of lung tumors induced by NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] by 69% and 75%. In animals exposed for 5 mo, 6 h/day, 5 days/wk, to a mixture of tobacco sidestream and mainstream smoke, and then fed myoinositol-containing diets once smoke exposure had ceased, no chemopreventive effect was observed. When animals were fed myoinositol during both tobacco smoke exposure and the recovery period, a slight but statistically not significant reduction in tumor multiplicities was found. It was concluded that myoinositol has less chemopreventive activity against the full complex mixture of tobacco smoke than it has against selected constituents such as NNK or benzo[a]pyrene.

GABA transmission in the ventral pallidum is not involved in the control of latent inhibition in the rat

Latent inhibition describes a process of learning to ignore stimuli of no consequence, and is disrupted in acute, positive-symptomatic schizophrenia. Understanding the neural basis of latent inhibition in animals may help to elucidate the neural dysfunction underlying positive schizophrenic symptoms in man. Evidence suggests a crucial role for dopamine transmission in the nucleus accumbens in the control of latent inhibition. The present studies investigated the role of the GABA-ergic efferent from the nucleus accumbens to the ventral pallidum in latent inhibition. The GABA A agonist muscimol (4.56 ng/μl), and antagonist picrotoxin (0.2 μg/μl), were infused into the ventral pallidum, and effects on latent inhibition were assessed using a conditioned suppression procedure. Neither drug produced specific effects on latent inhibition when given alone and, in the case of muscimol, failed to reverse the disruption of latent inhibition induced by systemic amphetamine. In addition to significant non-specific drug effects, a positive control experiment revealed that intra-pallidal picrotoxin significantly enhanced locomotion, suggesting that our manipulations of ventral pallidal GABA function were behaviourally effective. We conclude that modulating ventral pallidal GABA transmission does not affect latent inhibition. The implications of this finding for theories of the neural circuitry mediating latent inhibition and for understanding the functional role of ventral pallidal GABA transmission are discussed.